Poly-β-peptides from functionalized β-lactam monomers and antibacterial compositions containing same

ABSTRACT

Disclosed is a method of making β-polypeptides. The method includes polymerizing β-lactam-containing monomers in the presence of a base initiator and a co-initiator which is not a metal-containing molecule to yield the product β-polypeptides. Specifically disclosed are methods wherein the base initiator is potassium t-butoxide, lithium bis(trimethylsilyl)amide (LiN(TMS)2), potassium bis(trimethyl-silyl)amide, and sodium ethoxide, and the reaction is carried out in a solvent such as chloroform, dichloromethane, dimethylsulfoxide, or tetrahydrofuran.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 14/804,619,filed 21 Jul. 2015, issued as U.S. Pat. No. 9,683,081, which is acontinuation of application Ser. No. 13/075,218, filed 30 Mar. 2011,issued as U.S. Pat. No. 9,120,892, which is a divisional of applicationSer. No. 11/510,017, filed 25 Aug. 2006, issued as U.S. Pat. No.7,951,912, which claims priority to provisional application Ser. No.60/712,214, filed 29 Aug. 2005, and provisional application Ser. No.60/711,977, filed 26 Aug. 2005, all of which are incorporated herein byreference.

FEDERAL FUNDING STATEMENT

This invention was made with government support under 0425880 awarded bythe National Science Foundation. The government has certain rights inthe invention.

BACKGROUND

In recent years, a number of academic research groups have directedtheir interests toward oligomers and polymers comprised of β-amino acidresidues. These compounds are collectively referred to herein asβ-peptides or β-polypeptides. β-polypeptides differ from naturallyoccurring α-polypeptides by the presence of an additional carbon atom(the β-carbon) situated between the amino terminus and the carboxyterminus in the backbone of the polypeptide.

Over the past decade, the synthesis, properties and functions ofβ-peptides have been the subject of extensive study by a number ofresearch groups. See, for example, Cheng. Gelhman & DeGrado (2001) Chem.Rev. 101:3219-3232. The interest in β-polypeptides is due, in part, tothe discovery that these compounds can adopt stable secondary structures(“foldamers”) that mimic natural peptides. See Gellman et al. (1998)Acc. Chem. Res. 31:173. Certain β-polypeptides have been shown toexhibit important biological activity, including cholesterol absorptioninhibition and antimicrobial activity. Oligo-β-peptides generally mustbe prepared via solid-phase synthesis, a relatively costly andlabor-intensive technique that restricts the scope and scale of possibleuses for β-polypeptides, despite their favorable activities.

β-peptides are entirely non-natural. Thus, β-peptides are resistant toenzymatic degradation, in contrast to α-peptides. In short, β-peptidesmimic α-peptides in many key aspects (most notably the adoption ofstable secondary conformation) but because they are non-natural,β-peptides are not as prone to breakdown in biological milieus as arenaturally occurring α-peptides.

Large-scale synthesis of β-peptides is difficult because the standardpreparative methods involve step-wise, residue-by-residue synthesis.Thus, much of the scientific literature relating to β-polypeptidesapproaches the subject as a direct spin-off from α-polypeptidechemistry: The β-polypeptides are synthesized by solution-phase orsolid-phase chemistries, residue-by-residue. In the U.S. patentliterature, see, for example, U.S. Pat. Nos. 6,060,585; 6,613,876; and6,683,154, all to Gellman et al. See also U.S. Pat. No. 6,617,425, toSeebach. In the scientific literature, see, for example, Gellman et al.(2004) Organic Letters 6(24):4411-4; and Gellman et al. (1996) J. Am.Chem. Soc. 118:13071. See also Seebach et al. (1996) Helv. Chim. Acta.79:913-941; and Seebach et al. (1996) Helv. Chim. Acta. 79:2043-2066.Antibacterial compositions containing β-peptides are described in Hamuroet al. (1999)J. Am. Chem. Soc. 121:12200-12201.

Polymerization routes to β-peptides have been problematic mainly due tothe poor solubility of the resulting β-polypeptide chain, which limitsthe ability of the conventional routes to yield large β-polypeptideshaving a diverse number of derivatives. One route that has beeninvestigated by a number of groups is a ring-opening polymerization ofβ-lactams. See Graf et al. (1962) Angew. Chem. Int. Ed. 1:481; Sebendaet al. (1976) J. Polym Sci: Pol. Chem. 14:2357; Lopez-Carrasquero et al.(1994) Polymer 35:4502; Garcia-Alvarez et al. (1994) Syn. Commun.24:745; Hashimoto (2000) Prog. Polym. Sci. 25:1411; and Cheng et al.(2001) J. Am. Chem. Soc. 123:9457. Each of these routes, however,suffers from serious shortcomings related to low product solubility anddifficulty in characterizing the resulting products. Despite theirapparent convenience, the polymerization techniques described in thesereferences require high-purity reagents and solvents. Reactionconditions must be carefully controlled and maintained or thepolymerization falters. The conventional reactions as taught in thecited references simply are intolerant to impurities. The solventsystems that can be employed are also limited. Sebenda et al. franklystated that “It is known that polymers of β-lactams are only soluble instrongly polar solvents which interfere with anionic polymerization.” Inother words, the solvents required to keep the growing polymer chain insolution are solvents that are not conducive to anionic polymerization.

The patent literature likewise contains a number of issued U.S. patentsdirected to various aspects of lactam polymerization. See, for example,Deming et al., U.S. Pat. No. 6,818,732. See also U.S. Pat. Nos.6,881,819; 6,835,774; 6,013,758; 5,864,007, 5,756,647, 4,695,611; and4,677,189.

Thus, there remains a long-felt and unmet need for a synthetic route tomake large β-peptides (e.g., about 1 kDa or larger) that can becontrolled to yield homopolymers, random co-polymers, block copolymers,etc. of a desired molecular weight range and distribution.

SUMMARY OF THE INVENTION

Thus, the invention is directed to a method of making β-polypeptides.The method comprising polymerizing β-lactam-containing monomers in anorganic solvent in the presence of a base initiator and a co-initiatorwhich is not a metal-containing molecule. It is generally preferred thatthe co-initiator is a compound of formula

wherein X is independently a leaving group and R is independently abase-stable moiety, or R and X, together with the carbon atom to whichthey are attached, define a substituted or unsubstituted, five- totwelve-membered ring having an intramolecular leaving group. Preferablythe ring is monocyclic. When R and X are combined, the resulting ringmay be fully saturated, or may contain one or more unsaturations. Thering may also contain one or more heterocyclic atoms, generally selectedfrom N, O and S. For example, the co-initiator can be a cyclicanhydride, such as succinic anhydride or maleic anhydride. Theco-initiator can also be selected from the group consisting of:

wherein X is a leaving group selected from the group consisting ofalkoxide, amidate, carboxylate, halide, imidazolate, lactamate, andthiolate; and R is a base-stable moiety selected from the groupconsisting of linear, branched, or cyclic alkyl, alkenyl, alkynyl,substituted or unsubstituted aryl, and substituted or unsubstitutedarylalkyl, provided that the co-initiator is not an N-acyl-β-lactam.

In another version of the invention, at least one of theβ-lactam-containing monomers comprises a fused bicyclic β-lactam moiety.

In yet another version of the invention, the method uses at least oneβ-lactam-containing monomer is selected from the group consisting of:

wherein A, together with the carbon atoms to which it is attached isselected from the group consisting of substituted or unsubstitutedC₅-C₁₂ cycloalkyl, C₅-C₁₂ cycloalkenyl, and five- to twelve-memberedheterocyclic; and R³, R⁴, R⁵, and R⁶ are each independently selectedfrom the group consisting of hydrogen, substituted or unsubstitutedC₁-C₆-alkyl, aryl, C₁-C₆-alkylaryl, amino, protected-amino,amino-C₁-C₆-alkyl, and protected-amino-C₁-C₆-alkyl. In a preferred routeto yield β-polypeptides having an amino sidechain, at least one of R³,R⁴, R⁵, or R⁶ is selected from the group consisting of amino, protectedamino, amino-C₁-C₆-alkyl and protected amino-C₁-C₆-alkyl.

The base initiator is generally selected from the group consisting ofKOtBu, LiN(TMS)₂, KN(TMS)₂, NaOEt, Sc(N(TMS)₂)₃, Al₂(N(Me)₂)₆,Al(N(TMS)₂)₃, Zn(N(TMS)₂)₂, Sn(N(TMS)₂)₂, and CpTi(N(Me)₂)₂Cl. This listis non-limiting and other base initiators can be used.

The preferred solvents in which to carry out the method are chloroform,dichloromethane, dimethylsulfoxide, and tetrahydrofuran. Other suitablesolvents and mixed solvent systems may also be used.

The polymerization may be carried out using at least two differentβ-lactam-containing monomers to yield a co-polymer (e.g., a randomco-polymer or a block co-polymers)

In the preferred version, the polymerization reaction is carried out ata temperature≤about 50° C., preferably ≤about 30° C.

Another version of the invention is directed to a method of makingβ-polypeptides comprising: polymerizing bicyclic, β-lactam-containingmonomers in the presence of a base initiator and a co-initiator which isnot a metal-containing molecule, wherein the bicyclic,β-lactam-containing monomers are selected from the group consisting of:

wherein A, together with the carbon atoms to which it is attached isselected from the group consisting of substituted or unsubstitutedC₅-C₁₂ cycloalkyl, C₅-C₁₂ cycloalkenyl, and five- to twelve-memberedheterocyclic. The co-initiators and other reaction conditions are asnoted in the preceding paragraphs.

Another version of the invention is a method of making β-polypeptidescomprising polymerizing β-lactam-containing monomers in the presence ofa base initiator and a co-initiator which is not a metal-containingmolecule, wherein the β-lactam-containing monomers include a monomerselected from the group consisting of:

wherein R³, R⁴, R⁵, and R⁶ are each independently selected from thegroup consisting of hydrogen, substituted or unsubstituted C₁-C₆-alkyl,aryl, C₁-C₆-alkylaryl, amino, protected-amino, amino-C₁-C₆-alkyl, andprotected-amino-C₁-C₆-alkyl; and wherein at least one of R³, R⁴, R⁵, orR⁶ is selected from the group consisting of amino, protected amino,amino-C₁-C₆-alkyl, and protected amino-C₁-C₆-alkyl. This approach yieldsa β-polypeptide product having an amino side-chain.

Another version of the invention is directed to a method of makingβ-polypeptides comprising polymerizing monomers comprising substitutedβ-lactam-containing moieties, including at least one bicyclicβ-lactam-containing monomer, in an organic solvent, in the presence of abase initiator and a co-initiator which is not a metal-containingmolecule, at a temperature≤about 50° C., wherein:

the solvent is selected from the group consisting of chloroform,dichloromethane, dimethylsulfoxide, and tetrahydrofuran;

the base initiator is selected from the group consisting of KOtBu,LiN(TMS)₂, KN(TMS)₂, NaOEt, Sc(N(TMS)₂)₃, Al₂(N(Me)₂)₆, Al(N(TMS)₂)₃,Zn(N(TMS)₂)₂, Sn(N(TMS)₂)₂, and CpTi(N(Me)₂)₂Cl; and

the co-initiator is selected from the group consisting of:

wherein X is a leaving group selected from the group consisting ofalkoxide, amidate, carboxylate, halide, imidazolate, lactamate, andthiolate; and

R is a base-stable moiety selected from the group consisting of linear,branched, or cyclic alkyl, alkenyl, alkynyl, substituted orunsubstituted aryl, and substituted or unsubstituted arylalkyl, and

provided that the co-initiator is not an N-acyl-β-lactam.

Yet another version of the invention is directed to a method of makingβ-polypeptides comprising polymerizing monomers comprising substitutedβ-lactam-containing moieties, including at least one bicyclicβ-lactam-containing monomer, in an organic solvent, in the presence of abase initiator and a co-initiator which is not a metal-containingmolecule, at a temperature≤about 50° C., wherein:

the solvent is selected from the group consisting of chloroform,dichloromethane, dimethylsulfoxide, and tetrahydrofuran:

the base initiator is selected from the group consisting of KOtBu,LiN(TMS)₂, KN(TMS)₂, NaOEt, Sc(N(TMS)₂)₃, Al₂(N(Me)₂)₆, Al(N(TMS)₂)₃,Zn(N(TMS)₂)₂, Sn(N(TMS)₂)₂, and CpTi(N(Me)₂)₂Cl; and

the co-initiator is selected from the group consisting of:

wherein X is independently a leaving group and R is independently abase-stable moiety, or R and X, together with the carbon atom to whichthey are attached, define a substituted or unsubstituted, five- totwelve-membered ring having an intramolecular leaving group.

A distinct advantage of the present invention is that it allowsβ-polypeptides of large molecular weight (up to about 20,000 Da) to besynthesized in large quantities, quickly, with great control over thepolymerization conditions and the resulting product.

Another distinct advantage of the invention is that it allows for thepreparation of β-polypeptides containing cationic side-chains or groupsthat can be convened into cationic side-chains after polymerization. Forexample, the inventive method can be used to make β-polypeptides havingan amino moiety or a tBOC-protected amino moiety as a side-chain. Thesenitrogen-containing groups that can subsequently be manipulated (e.g.quaternized or otherwise further functionalized after polymerization).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a gel permeation chromatography curve for the polymer formedfrom compound 3.

FIG. 2 depicts superimposed gel permeation chromatography curves of ahomopolymer (right-hand peak) and a diblock co-polymer (left-hand peak)made according to the present invention.

FIG. 3 is graph depicting the linear dependence of product molecularweight (M_(n)) versus the ratio of monomer concentration to co-initiatorconcentration ([monomer]/[co-initiator]) for the polymerization ofcompound 6.

FIG. 4 is a gel permeation chromatography curve of a homopolymer madeusing compound 6.

FIG. 5 is a gel permeation chromatogram of degenerate block co-polymermade using compound 6.

DETAILED DESCRIPTION

Abbreviations and Definitions

The following abbreviations and definitions are used throughout thespecification and claims. Terms not given an explicit definition hereare to be given their art-accepted definition in the field of organicchemistry.

Amino=refers to a chemical group or moiety containing an sp² or sp³hybridized nitrogen atom, e.g., mono- and di-substituted amines wherethe nitrogen atom is in an sp³ hybridization state, and pyridine andimidazole, where the nitrogen is in an sp2 hybridization state.

β-Lactam=azetidin-2-one:

β-Lactam-containing monomer=a polymerizable monomer that comprises aβ-lactam moiety. β-lactam itself is a “β-lactam-containing monomer.”

Et=ethyl.

GPC=gel permeation chromatography.

KN(TMS)₂=potassium bis(trimethylsilyl)amide.

KOtBo=potassium-tert-butoxide.

Lactamate=a moiety of the formula:

wherein Y is a substituted or unsubstituted C₃- to C₁₁-alkylene,alkenylene, or alkynylene. Y in combination with the nitrogen atom andthe carbonyl carbon to which it is attached may define a monocyclic orbicyclic moiety. The monocyclic or bicyclic moiety may be unsubstitutedor substituted with one or more of halo, alkyl, aryl (e.g., phenyl),halo-substituted aryl, and/or alkyl-substituted aryl. The unqualifiedterm “lactam” refers to the corresponding neutral molecule wherein thenitrogen atom is bonded to a hydrogen.

Leaving group=a labile atom or moiety that becomes detached from theco-initiator to yield a corresponding anion. As used herein, the term“leaving group” explicitly includes, without limitation, alkoxides,amidates, carboxylates, halides, imidazolates, lactamates, thiolates,and the like.

LiN(TMS)₂=lithium bis(trimethylsilyl)amide.

Me=methyl.

MeONa=sodium methoxide.

PDI=polydispersity index.

Phth=phthalimide.

Protecting Group/Protected Amine=A “protecting group” refers to achemical moiety that exhibits the following characteristics:) reactsselectively with the desired functionality in good yield (preferably atleast 80%, more preferably at least 90%, more preferably at least 95%,still more preferably at least 99%) to give a protected substrate thatis stable to the projected reactions for which protection is desired; 2)is selectively removable from the protected substrate to yield thedesired functionality; and 3) is removable in good yield (preferably atleast 80%, more preferably at least 90%, more preferably at least 95%,still more preferably at least 99%) by reagents compatible with theother functional group(s) present or generated in such projectedreactions. Carbamate-, sulfonamide-, sulfamate-, and ammonium-formingprotecting groups may all be used. Because the polymerization reactionoccurs under basic conditions, base-stable protecting groups arepreferred. The term “protecting group” explicitly includes, withoutlimitation t-butoxycarbonyl (tBOC), benzyloxycarbonyl (Cbz), benzyl(Bn), and allyloxycarbonyl (alloc). A “protected amine” is an aminemoiety protected by a “protecting group.” A host of suitableamine-protecting groups are known in the art. See, for example, Greene &Wuts, “Protective Groups in Organic Synthesis, Third Edition.” © 1999,Wiley-Interscience/John Wiley & Sons, New York, N.Y. (ISBN0-471-16019-9).

Substituted or unsubstituted=when referring to a chemical moiety, thephrase “substituted or unsubstituted” means that the chemical moiety mayappear as the basic unsubstituted moiety (e.g., an alkyl group having noother molecules beyond carbon and hydrogen), or the chemical moiety issubstituted with one or more substituents, e.g. alkyl, halogen, alkoxy,acyloxy, amino, hydroxy, mercapto, carboxy, benzyl, etc.

TBBC=4-tert-butyl benzoyl chloride.

tBOC=tert-butoxycarbonyl.

Chemistry:

To overcome the limitations noted earlier, the present inventors havedeveloped a synthetic route for fabricating β-peptides via aring-opening polymerization of β-lactam-containing monomers. Exemplaryreactions are shown in Reaction Schemes 1 and 2:

Note that the reactant monomers can be monocyclic (e.g., see monomers 10and 13 in the Examples) or bicyclic (e.g., see Reaction Schemes 1 and 2,which illustrate the polymerization of compound 6; see the Examples). Asshown in Reaction Schemes 1 and 2, the reaction proceeds in the presenceof a base (potassium-t-butoxide in these two representative reactions)and a co-initiator (N-benzoyl-4-phenyl-β-lactam, also knownsystematically as N-benzoyl-4-phenyl azetidin-2-one).

The inventive reaction route is extremely versatile and has a great manybenefits. Most notably, β-peptides of controlled molecular weights (Mn)of anywhere from about 1,000 Da to about 20,000 and larger can beobtained, using very common (and cheap) reagents. In typical reactions(where the time of reaction is not being unduly extended), β-peptides ofcontrolled molecular weights of from about 1,000 Da to about 12,000 Daare readily obtained. Many of the β-polypeptide polymers thus obtainedare soluble in common organic solvents, including dichloromethane,chloroform, and tetrahydrofuran (THF). The molecular weight distributionof the resulting polymers is quite narrow. For example, the monomershown in Reaction Scheme 1 (a bicyclic compound comprising a β-lactamring fused to a cyclooctane ring) was polymerized according to thepresent invention via an opening of the β-lactam ring to yield a polymerhaving an Mn of 11,400 Da (as measured by gel permeation chromatography;see FIG. 1), with a polydispersity index (PDI, calculated via gelpermeation chromatography using a laser light-scattering detectors) ofonly 1.05. (“Polydispersity index” is also sometimes referred to as“molecular weight distribution” and is the ratio of the weight-averagemolecular weight (Mw) to number-average (Mn) molecular weight.) As ageneral rule, the inventive method will generate β-polypeptides with aPDI of less than about 2.0, more preferably less than about 1.5, andmore preferably still less than about 1.3.

The reaction can proceed either in the presence or absence of aco-initiator. The polymerization reaction will proceed without anco-initiator, but the PDI tends to rise without the co-initiator.Suitable non-β-lactam co-initiators include aromatic acyl halides,preferably substituted or unsubstituted benzoyl halides, such as4-tert-butyl-benzoyl chloride and 4-chloromethyl benzoyl chloride (bothof which can be obtained commercially from several internationalsuppliers, including Sigma-Aldrich, Milwaukee, Wis.), and the like.

As shown in Reaction Scheme 2, the reaction is a living polymerizationand thus can be used to fabricate homopolymers, random co-polymers,block co-polymers, and the like. As used herein, the term “livingpolymerization” assumes its conventional meaning in the art, namely: apolymerization in which the ability of a growing polymer chain toterminate has been inhibited or abolished. Thus, the polymerizationreaction can be carried out in stages, using monomers, first-stagepre-polymers, and/or second-stage (and/or subsequent-stage) pre-polymersas the reactants. Because the reaction is a living polymerization, theinventive route described herein provides exquisite control of thepolymerization process.

The reactions shown in Reaction Scheme 2 and resulting GPC curves (seeFIG. 2) demonstrate the ability of the present invention to fabricateco-polymers. Moving left-to-right across Reaction Scheme 2, in a firstreaction, a β-polypeptide homopolymer was fabricated from compound 6(see the Examples). The resulting homopolymer, shown in the middle ofReaction Scheme 2 had a molecular weight (Mn) of 2,500, and a PDI of1.13). In FIG. 2, the resulting GPC curve for the homopolymer is theright-hand peak.

Alternatively, a reactive terminal end-group can be used for terminalfunctionalization of the polymer or a different monomer can beintroduced to yield a co-polymer. As shown in the right-hand portion ofReaction Scheme 2, additional monomer was added to the on-goingpolymerization to alter the ultimate molecular weight of the resultingpolymer. The GPC curve of the resulting “m+n” co-polymer is theleft-hand peak in FIG. 2. The “m+n” co-polymer shown in Reaction Scheme2 had the following characteristics: Mn=17,300, PDI=1.23. The increasedmolecular weight of the “m+n” co-polymer of Reaction Scheme 2 ascompared to the homopolymer of Reaction Scheme 2 is readily apparent bythe leftward shift of the GPC peak of the co-polymer as compared to theGPC peak for the homopolymer. The comparability of the PDIs is alsoapparent as evidenced by the widths of the two peaks, which are quitesimilar (PDI=1.13 for the homopolymer, 1.23 for the co-polymer). Thesedata are significant because they demonstrate that the present inventioncan be used to fabricate β-peptides of vastly different molecularweights (homopolymers, co-polymers, and terminally-functionalizedpolymers) in a controlled fashion (and without significantly increasingthe polydispersity of the resulting polymers). In short, the exemplaryreaction depicted in Reaction Scheme 2 yields a relatively smallhomopolymer and a comparatively far larger co-polymer, yet the PDI's forboth products are very similar. See the Examples for a furtherdiscussion.

The present inventive reaction is both highly flexible and robust.Unlike past approaches, which are very sensitive to solvent effects andimpurities, the present reaction will proceed using a host of low-costinitiators and solvents. The reaction is also robust and tolerant ofimpurities.

For example, polymerization of cyclooctyl-β-lactam was also tested inthe presence of up to 20% mol of water or benzyl amine. The molecularweight and the PDI of the resulting polymers were unaffected relative toanalogous reactions without the added water or benzyl amine. See Table2. In the polymerization of compound 6, using the method of the presentinvention, product having a PDI less than 1.5 were obtained under a hostof less-than-ideal conditions. The general reaction is shown in ReactionScheme 3:

β-polypeptides were fabricated according to Reaction Scheme 3 usingbicyclic β-lactam ring-opening anionic polymerization in common solventsincluding dichloromethane, tetrahydrofuran, and dimethylsulfoxide. Thereaction can be initiated using common base initiators, including(without limitation) KOtBu, UN(TMS)₂, and MeONa (in tetrahydrofuran).

Another distinct advantage of the present invention is that it allows ahost of functional groups to be incorporated into the resulting polymer(either during the polymerization itself or via subsequent reactionsinvolving reactive side groups post-polymerization.) For example, theβ-lactam monomers can include functional groups on the side chains thatare hydrophilic, hydrophobic, anionic, cationic, etc. The ratios ofthese various side chains within the final polymer can be controlled bycontrolling the relative amounts of each monomer in theco-polymerization reaction. The hydrophobic and cationic co-polymers,which are shown in the Examples, are particularly noteworthy becausethese side chains contribute to the antibacterial functionality of thepolymers. Of course, the side chains can be manipulated to optimize anyother desired property of the resulting polymer, be it solubility,biological activity, etc.

Additional monocyclic and bicyclic β-lactam monomers that have beenfabricated and polymerized are shown in Reaction Scheme 4 (bicyclicmonomers) and Reaction Scheme 5 (monocyclic monomers):

Reaction Scheme 4 illustrates the polymerization of bicyclic β-lactammonomers. Shown in the Reaction Scheme 4 are monomers that include afused cyclooctene ring and a fused cyclododecane ring. Reaction Scheme 5illustrates the polymerization of di- and tri-substituted monocyclicβ-lactam monomers. The resulting polymers shown in Reaction Schemes 4and 5 are obtained in high-yield (>90%), with very low molecular weightdistributions (PDI's<1.5). See the Examples for further details.

Not all β-lactam-containing monomers will yield soluble products. Note,however, that the invention explicitly encompasses methods that yieldsoluble or insoluble polymeric products. For example, the following twoβ-lactam-containing monomers yield insoluble polymers when polymerizedaccording to the present invention:

(See the Examples for a complete recitation of the reaction specifics.)Of particular note, however, is that all of these monomers can bereadily polymerized in CH₂Cl₂ or THF, and the resulting polymers havevery small PDIs, generally <1.5.

The synthetic route described herein is highly useful because a numberof β-peptides and related compounds have been shown to be antimicrobial.See, for example, the Gellman et al. patents noted in the Backgroundsection. Thus, the present inventive method provides a new and robustroute to making large quantities of β-peptides for medicinal use.

Additionally, the present invention is useful because it provides aversatile method to polymerize β-lactam-containing monomers undercontrolled conditions. The resulting polymers can then be used forsystematic probing of a large array of polymer structures. For example,the present method allows systematic fabrication of homopolymers ofknown molecular weight and polydispersity. The method also allows forthe systematic fabrication of random and block co-polymers usingdifferent combinations of co-monomers, monocyclic and bicyclic,including, without limitation, the following β-lactam-containingmonomers:

As a result, a whole host of β-peptides can be fabricatedsystematically, in molecular weights up to and greater than 20 kDa,including homopolymers, for example:

random co-polymers, for example:

and block co-polymers, for example:

Further still, because the present method provides a systematic route tofabricating β-peptides, it also provides a robust means to optimizedesired biological activities of β-peptides. In the past, this couldonly be done via step-wise, residue-by-residue synthesis—aextraordinarily laborious and time-consuming approach. In contrast, forexample, the present method was used to synthesize a series of β-peptidehomopolymers and co-polymers, which were then tested for antibacterialactivity. Selected examples are shown in Table 1:

TABLE 1 Minimum Inhibitory Concentrations and Hemolytic Concentrations(HC₅₀)

Min. Inhibitory Conc. (MIC) Against: E. coli 200 μg/mL 100 μg/mL 25μg/mL B. subtilis 12.5 μg/mL 6.25 μg/mL 6.25 μg/mL S. aureus 12.5 μg/mL25 μg/mL 50 μg/mL Conc for 50% lysis of >1000 μg/mL 100 μg/mL n/a humanred blood cells (HC₅₀)As can be seen from Table 1, the left-most polymer exhibitedantimicrobial activity against E. coli, B. subtilis, and S. aureus, withMIC values of 200 μg/mL, 12.5 μg/mL, and 12.5 μg/mL, respectively, whileat the same time exhibiting a vastly higher hemolytic concentration(HC₅₀), greater than 1,000 μg/mL In other words, at concentrations wherethis compound is effective to inhibit the growth of E. coli, B.subtilis, and S. aureus, it exhibits very little hemolysis. (See theExamples for complete experimental details on how the values obtained inTable 1 were generated.)

Thus, the present invention can be used to synthesize β-polypeptidesthat have desirable biological properties, such as antimicrobialactivity. The invention is, in effect, an general purpose and robustmethod to synthesize large quantities of β-polypeptides under mild andcontrollable conditions.

If the ultimate product is to be incorporated into a pharmaceuticalcomposition, the composition is preferably formulated by means generallyknown the industry. Thus, pharmaceutical compositions according to thepresent invention comprise an effective amount of a β-aminoacid-containing polypeptide or a pharmaceutically acceptable saltthereof together with a pharmaceutically acceptable carrier. Optionally,other therapeutically active substances or accessory agents may beincluded in addition to the β-polypeptide or the salt thereof. Thepharmaceutical compositions of the invention comprise an amount ofβ-polypeptide or a pharmaceutically acceptable salt thereof that iseffective to treat a bacterial, viral, or fungal infection in a mammalsuffering therefrom (including humans). The carrier must bepharmaceutically acceptable in the sense of being compatible with theother ingredients in the particular composition and not deleterious tothe recipient of the composition. The compositions include thosesuitable for oral, topical, rectal or parenteral (includingsubcutaneous, intramuscular, intradermal and intravenous)administration.

In a particular aspect, the pharmaceutical compositions comprise theactive ingredient (a β-polypeptide or a pharmaceutically acceptable saltthereof) presented in unit dosage form. The term “unit dosage” or “unitdose” designates a predetermined amount of the active ingredientsufficient to be effective to treat each of the indicated activities.Preferred unit dosage formulations are those containing a daily dose,daily sub-dose, or an appropriate fraction thereof, of the administeredactive ingredient.

The pharmaceutical compositions may be prepared by any of the methodswell known in the art of pharmacy. All methods include the step ofbringing the active ingredient into association with a carrier whichconstitutes one or more accessory ingredients. In general, thecompositions are prepared by uniformly and intimately bringing theactive ingredient into association with a liquid or solid carrier andthen, if necessary, shaping the product into the desired unit dosageform.

Compositions of the present invention suitable for oral administrationmay be presented as discrete unit dosages, e.g., as capsules, cachets,tablets, boluses, lozenges and the like, each containing a predeterminedamount of the active ingredient; as a powder or granules; or in liquidform, e.g., as a collyrium, suspension, solution, syrup, elixir,emulsion, dispersion and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active compound in a free-flowingform, e.g., a powder or granules, optionally mixed with accessoryingredients or excipients, e.g., binders, lubricants, inert diluents,surface-active or dispersing agents. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered active compoundwith any suitable carrier.

Compositions suitable for parenteral administration convenientlycomprise a sterile injectable preparation of the active ingredient in,for example, a solution which is preferably isotonic with the blood ofthe recipient. Useful formulations also comprise concentrated solutionsor solids containing the active ingredient which upon dilution with anappropriate solvent give a solution suitable for parenteraladministration. The parenteral compositions include aqueous andnon-aqueous formulations which may contain conventional adjuvants suchas buffers, bacteriostats, sugars, thickening agents and the like. Thecompositions may be presented in unit dose or multi-dose containers, forexample, sealed ampules and vials.

Compositions suitable for topical or local application (includingophthamological administration) comprise the active ingredientformulated into pharmaceutically-acceptable topical vehicles byconventional methodologies. Common formulations include drops,collyriums, aerosol sprays, lotions, gels, ointments, plasters,shampoos, transferosomes, liposomes and the like.

Compositions suitable for inhalation administration, for example, fortreating bronchial infections, wherein the carrier is a solid, include amicronized powder or liquid formulation having a particle size in therange of from about 5 microns or less to about 500 microns, for rapidinhalation through the nasal or oral passage from a conventionalinhalation squeeze or spray container. Suitable liquid nasalcompositions include conventional nasal sprays, nasal drops and thelike, of aqueous solutions of the active ingredient and optionaladjuvants.

In addition to the aforementioned ingredients, the compositions of thisinvention may further include one or more optional accessoryingredients(s) utilized in the art of pharmaceutical formulations, e.g.,diluents, buffers, flavoring agents, colorants, binders, surfactants,thickeners, lubricants, suspending agents, preservatives (includingantioxidants) and the like.

The amount of active ingredient required to be effective for each of theindicated activities will, of course, vary with the individual mammalbeing treated and is ultimately at the discretion of the medical orveterinary practitioner. The factors to be considered include thespecies and sex of the mammal, the ailment being treated, the route ofadministration, the nature of the formulation, the mammal's body weight,surface area, age and general condition, and the particular compound tobe administered.

In general, the pharmaceutical compositions of this invention containfrom about 0.5 to about 500 mg and, preferably, from about 5 to about350 mg of the active ingredient, preferably in a unit dosage form, foreach of the indicated activities. However, a suitable effective dose isin the range of about 0.1 to about 200 mg/kg body weight per day,preferably in the range of about 1 to about 100 mg/kg per day,calculated as the non-salt form of the β-polypeptide. The total dailydose may be given as a single dose, multiple doses, e.g., two to sixtimes per day, or by intravenous infusion for a selected duration.Dosages above or below the range cited above are within the scope of thepresent invention and may be administered to the individual patient ifdesired and necessary.

For example, for a 75 kg mammal, a dose range would be about 7.5 toabout 1500 mg per day, and a typical dose would be about 800 mg per day.If discrete multiple doses are indicated, treatment might typically be200 mg of the active agent, twice per day.

In topical formulations, the subject compounds are preferably utilizedat concentrations of from about 0.1% to about 5.0% by weight.

EXAMPLES

The following Examples are included to provide a more completeunderstanding of the invention disclosed and claimed herein. TheExamples do not limit the scope of the invention in any fashion.

Example 1—Monomer and Co-Initiator Synthesis

Synthesis of Coinitiator (2):

Compound 1 was prepared using the method of Huang and coworkers (Huang,H.; Iwasawa, N.; Mukaiyama, T. (1984) “A Convenient Method for theConstruction of β-Lactam Compounds from β-Amino Acids Using2-Chloro-1-Methyl Pyridinium Iodide as Condensing Reagent,” Chem. Lett.1465-1466). In a 1 L round-bottomed flask was combinedDL-3-amino-3-phenyl propionic acid (0.007 mol, 1.156 g),2-chloro-1-methyl pyridinium iodide (1.1 eq., 0.0077 mol, 1.74 g),acetonitrile (700 mL) and triethylamine (2.2 eq., 0.0154 mol, 2.15 mL).The reaction was stirred under nitrogen and heated to reflux overnight.The solvent was removed by rotary evaporation and the crude product waspurified by column chromatography in 1:1 hexanes: ethyl acetate. Yield:0.808 g, 40%. ¹H NMR (CDCl₃) δ 2.84-2.9, m, 1H; 3.40-3.48, m, 1H;4.71-4.74, m, 1H; 6.38, br s, 1H; 7.2-7.4, m, 5H.

Compound 2 was prepared using the method of Park and coworkers (Park.H.; Hepperle, M.; Boge, T. C.; Himes, R. H.; Georg, G. I. (1996)“Preparation of Phenolic Paclitaxel Metabolites,” J. Med. Chem.39:2705-2709). In a 25 mL round-bottomed flask was combined (1) (0.0017mol, 0.250 g), triethylamine (4.63 eq., 0.0073 mol, 1.02 mL), drymethylene chloride (6.3 mL), and dimethylamino pyridine (10 mol %, 1.7E-4 mol, 0.021 g). The reaction was cooled to 0° C. and benzoyl chloride(3.33 eq., 0.0057 mol, 0.66 mL) was added. The reaction was warmed toroom temperature and stirred for 1 h. The reaction was quenched withsaturated ammonium chloride (30 mL) and diluted with methylene chloride(150 mL). The reaction mixture was then washed with NaHCO₃ and then withbrine. The organic portion was dried over MgSO₄ and the solvent removedby rotary evaporation. The crude product was purified by columnchromatography in 1:1 hexanes: ethyl acetate. Yield: 0.320 g, 75%. ¹HNMR (CDCl₃) δ 3.094, dd J=16.5, 3.9 Hz, 1H; 3.528, dd J=16.5, 6.9 Hz,1H; 5.29, m, 1H; 7.26-7.60, m, 8H; 8.01-8.04, m, 2H. ¹³C {¹H} NMR(CDCl₃) δ 44.23, 51.56, 125.77, 127.99, 128.26, 128.73, 129.75, 131.68,133.22, 137.99, 163.91, 165.65. FTIR (ATR): 1675 cm⁻¹, 1735 cm⁻¹, 1778cm⁻¹, 1795 cm⁻¹. MS-ESI: m/z=525.2 [2 M+Na]⁺.

β-Lactam 3 was synthesized according to the literature precedent. SeeParsons, P. J.; Camp, N. P.; Underwood, J. M., Harvey, D. M. (1996)“Tandem Reactions of Anions: A Short and Efficient Route to±Anatoxin-a,” Tetrahedron, 52:11637-11642.

β-Lactams 4 and 5 were synthesized according to the literatureprecedent. See Dener. J. M.; Fantauzzi. P. P.; Kshirsagar, T. A.; Kelly.D. E.; Wolfe. A. B. (2001) “Large-Scale Synthesis of FMOC-ProtectedNon-Proteogenic Amino Acids: Useful Building Blocks for CombinatorialLibraries,” Organic Process Research and Development, 5:445-449.

Compounds 6 and 7 were prepared using the same general method used forcompounds 4 and 5.⁴ For compound 6: In an oven-dried 25 mLround-bottomed flask was combined cis-cyclooctene (0.023 mol, 3 mL) anddry CH₂Cl₂ (3.3 mL). The reaction was cooled to 0° C. and stirred underN₂. A solution of chlorosulfonyl isocyanate (CSI) (1 eq., 0.023 mol, 2mL) in dry CH₂Cl₂ (1.1 mL) was made and added dropwise to the cooledreaction mixture. The reaction was allowed to stir at 0° C. for 1 h andthen warned to room temperature overnight. The reaction was thenre-cooled to 0° C. and quenched by adding water. The quenched reactionmixture was added to a suspension of Na₂SO₃ (1.45 g) in water (4.3 mL),keeping the temperature below 25° C. and the pH between 5 and 7 using 2M NaOH. The reaction was allowed to warm to room temperature overnight.The layers were separated and the aqueous portion was extracted withEtOAc. The combined organic portions were dried over MgSO₄ and thesolvent removed by rotary evaporation. The crude product can be purifiedby column chromatography (EtOAc as eluent) or recrystallization fromCH₂Cl₂ and hexanes. Yield=3.6 g, 51%. ¹H NMR (CDCl₃) δ 1.30-1.99, m,12H; 3.01-3.10, m, 1H; 3.62-3.69, m, 1H; 5.86, br s, 1H, ¹³C {¹H} NMR(CDCl₃) δ 21.34, 25.25, 25.69, 27.24, 28.59, 27.72, 53.58, 171.0. FTIR(ATR): 1725 cm⁻¹, 3205 cm⁻¹. MS-EI: m/z=154.1 [M+H]⁺.

For Compound 7: In an oven-dried round-bottomed flask was combinedcyclododecene (0.023 mol, 3.97 mL) and CSI (1 eq., 0.023 mol, 2 mL). Thereaction was put under nitrogen and heated to 50° C. overnight. Thereaction was allowed to cool, diluted with CH₂Cl₂, and quenched byadding water. The quenched reaction was added to a suspension of Na₂SO₃(1.6 g) in water (5 mL), keeping the temperature below 25° C. and the pHbetween 5 and 7 using 2 M NaOH. The reaction was allowed to warm to roomtemperature overnight and then the layers were separated. The aqueouslayer was extracted twice with EtOAc and the combined organic layerswere dried over MgSO₄ and the solvent was removed by rotary evaporation.The crude product was purified by recrystallization from CH₂Cl₂ andhexanes. Yield=0.47 g, 10%. ¹H NMR (CDCl₃) δ 1.30-1.80, m, 20H;3.10-3.16, min, 1H; 3.64-3.70, m, 1H; 6.11, br s, 1H. ¹³C {¹H} NMR(CDCl₃) δ 2.24, 22.84, 23.22, 24.90, 27.62, 27.83, 28.12, 28.21, 29.20,53.20, 54.20, 171.78. FTIR (ATR): 1740 cm⁻¹, 3204 cm⁻¹. MS-ESI:m/z=232.3 [M+Na]⁺.

For Compound 8: In a 500 mL round-bottomed flask was combined potassiumphthalimide (1.5 eq., 0.067 mol, 12.33 g) and DMF (140 mL). The reactionwas stirred and a solution of 1-chloro-3-methyl-2-butene (1 eq., 0.044mol, 5 mL) in DMF (95 mL) was added to the reaction. The flask was putunder N₂ and heated to 60° C. overnight. The reaction was allowed tocool and then poured into 1400 mL ice and water with vigorous stirring.The stirring was continued until the ice melted. The resulting whiteprecipitate was isolated by filtration. The wet solid was dissolved inCH₂Cl₂ and the layers were separated. The organic portion was dried overMgSO₄ and the solvent removed by rotary evaporation to give crudeproduct. The product was purified by recrystallization from CH₂Cl₂ andhexanes. Yield=7.2 g, 76%.

Compound 9 was prepared from Compound 8 in the following manner: In a100 mL round-bottomed flask was placed Compound 8 (1 eq., 0.033 mol, 7g). It was dissolved in as little dry CH₂Cl₂ as possible and the flaskput under N₂ and cooled to 0° C. CSI (1 eq., 0.033 mol, 2.9 mL) wasadded to the flask and the reaction allowed to stir and warm to roomtemperature for 1-2 days. The reaction was quenched by adding water andthe quenched reaction mixture was added to a suspension of Na₂SO₃ andNa₂HPO₄ (5 g each) in water (140 mL). The pH was maintained between 5and 7 using 2 M NaOH and the reaction was allowed to stir at roomtemperature for 2 days. The layers were then separated and the aqueousportion was extracted twice with CH₂Cl₂. The combined organic portionswere dried over MgSO₄ and the solvent removed by rotary evaporation. Thecrude product was recrystallized from CH₂Cl₂ and hexanes. Yield=6.2 g,73%. ¹H NMR (CDCl₃) δ 1.45, s 3H; 1.47, s, 3H; 3.41, td J=8.1, 0.9 Hz,1H; 3.91, dd J=14.1, 8.1 Hz, 1H; 4.05-4.13, m, 1H; 6.44 br s, 1H;7.71-7.74, m, 2H; 7.84-7.87, m, 2H. ¹³C {¹H} NMR (CDCl₃) δ 14.31, 21.15,23.38, 25.58, 34.31, 55.18, 56.66, 60.48, 123.52, 132.11, 134.17,167.47, 168.06. FTIR (ATR): 1715 cm⁻¹, 1741 cm⁻¹, 3200 cm⁻¹. MS-ESI:m/z=259.3 [M+H]⁺.

Compound 10 was prepared from Compound 9 in the following manner: In a250 mL round-bottomed flask was suspended Compound 9 (1 eq., 0.024 mol,6.2 g) in methanol (36 mL). Hydrazine (3 eq., 0.072 mol, 2.26 mL) wasadded and the reaction was allowed to stir at room temperature under N₂overnight. The methanol was removed by rotary evaporation and theresulting solid was triturated with chloroform. The solvent was removedfrom the chloroform washings by rotary evaporation. The residue wasplaced in a 500 mL round-bottomed flask with CH₂Cl₂ (200 mL).Triethylamine (1.1 eq., 0.053 mol, 7.39 mL) was then added followed by asolution of di-tert-butyl-dicarbonate (BOC₂O) (1.1 eq., 0.053 mol, 11.6g) in CH₂Cl₂ (100 mL). The reaction was allowed to stir at roomtemperature overnight and then washed twice with 2 M HCl, twice with 2 MNaOH, and once with brine before being dried over MgSO₄ and stripped byrotary evaporation to yield crude product. The product was purified bycolumn chromatography using EtOAc as eluent. Yield=2.9 g, 53%. ¹H NMR(CDCl₃) 1.40, s, 3H; 1.44, s, 3H; 1.45, s, 9H; 2.97 app t J=7.8 Hz, 1H;3.29, m, 1H; 3.58, m, 1H; 5.10, br, 1H. ¹³C {¹H} NMR (CDCl₃) δ 22.92,28.49, 28.68, 37.20, 54.83, 58.34, 79.61, 155.89, 169.32. FTIR (ATR):1688 cm⁻¹, 1716 cm⁻¹, 1744 cm⁻¹, 3194 cm⁻¹, 3280 cm⁻¹. MS-ESI/EMM:m/z=Calc. 251.1372 [M+Na]⁺. Meas. 251.1372 [M+Na]⁺.

Compound 11: In a 2 L round-bottomed flask was combined potassiumphthalimide (1.5 eq., 0.28 mol, 52 g) and DMF (400 mL). A solution ofcrotyl bromide (1 eq., 0.185 mol, 25 g) in DMF (300 mL) was then addedand the reaction stirred at 60° C. overnight. The reaction was allowedto cool and then poured into 4000 mL ice and water with vigorousstirring. The stirring was continued until the ice melted. The resultingwhite precipitate was isolated by filtration. The wet solid wasdissolved in CH₂Cl₂ and the layers were separated. The organic portionwas dried over MgSO₄ and the solvent removed by rotary evaporation togive crude product. The product was purified by recrystallization fromCH₂Cl₂ and hexanes. Yield=17.8 g, 56%.

Compound 12: In a 100 mL round-bottomed flask was placed Compound 11 (1eq., 0.085 mol, 17 g). It was dissolved in as little dry CHCl₃ aspossible and the flask put under N₂ and cooled to 0° C. CSI (1 eq.,0.033 mol, 2.9 mL) was added to the flask and the reaction allowed tostir and warm to room temperature, then heated to 60° C. for 4-5 days.The reaction was quenched by adding water and the quenched reactionmixture was added to a suspension of Na₂SO₃ and Na₂HPO₄ (40 g each) inwater (700 mL). The pH1 was maintained between 5 and 7 using 2 M NaOHand the reaction was allowed to stir at room temperature for 2 days. Thelayers were then separated and the aqueous portion was extracted twicewith CH₂Cl₂. The combined organic portions were dried over MgSO₄ and thesolvent removed by rotary evaporation. The crude product wasrecrystallized from CH₂Cl₂ and hexanes. Yield=7.6 g, 38%. ¹H NMR (CDCl₃)δ 1.32, d J=6 Hz, 3H; 3.15 app t J=6.9 Hz, 1H; 3.79-3.83, m, 1H; 3.97,dd J=14, 9.6 Hz, 1H: 4.14, dd J=14, 5.7 Hz, 1H; 6.01, br s, 1H;7.72-7.77, m, 2H; 7.84-7.88, m, 2H. ¹³C {¹H} NMR (CDCl₃) δ 20.75, 36.62,50.46, 57.12, 123.69, 132.11, 134.37, 167.07, 168.23. MS-ESI: m/z=267.2[M+Na]⁺.

Compound 13: In a 25 mL round-bottomed flask was suspended Compound 12(1 eq., 0.0021 mol, 0.5 g) in methanol (10 mL). Hydrazine (5 eq., 0.0105mol, 0.33 mL) was added and the reaction was allowed to stir at roomtemperature under N₂ overnight. The reaction was filtered on a frit andwashed with copious amounts of methanol. The solvent was removed fromthe filtrate by rotary evaporation. The residue was placed in a 100 mLround-bottomed flask with CH₂Cl₂ (20 mL). Triethylamine (1.1 eq., 0.0057mol, 0.8 mL) was then added followed by a solution ofdi-tert-butyl-dicarbonate (BOC₂O) (1.1 eq., 0.0057 mol, 1.25 g) inCH₂Cl₂ (10 mL). The reaction was allowed to stir at mom temperatureovernight and then washed twice with 2 M HCl, twice with 2 M NaOH, andonce by brine before being dried over MgSO₄ and stripped by rotaryevaporation to yield crude product. The product was purified by columnchromatography using EtOAc as eluent. Yield=0.071 g, 16%. ¹H NMR (CDCl₃)δ 1.37, d J=6 Hz, 3H; 1.44, s, 9H; 2.89, tdd J=6, 2.1, 0.9 Hz, 1H; 3.48,m, 2H; 3.64, qd J=6, 2.1 Hz, 1H; 4.95, br s, 1H; 6.06, br s, 1H. ¹³C NMR(CDCl₃) δ20.54, 28.55, 38.46, 48.84, 58.57, 79.89, 164.71, 168.99.MS-ESI/EMM: m/z=Calc. 237.1215 [M+Na]⁺. Meas. 237.1208 [M+Na]⁺.

Compound 14: In a dry 25 mL round-bottomed flask was placedtrans-4-octene (1 eq., 0.009 mol, 1.4 mL). CSI (1 eq., 0.009 mol, 0.78mL) was added to the flask and the reaction allowed to stir at 60° C.overnight. The reaction was diluted with CH₂Cl₂ and then quenched byadding water. The quenched reaction mixture was added to a suspension ofNa₂SO₃ and Na₂HPO₄ (1 g each) in water (20 mL). The pH was maintainedbetween 5 and 7 using 2 M NaOH and the reaction was allowed to stir atroom temperature overnight. The layers were then separated and theaqueous portion was extracted twice with CH₂Cl₂. The combined organicportions were dried over MgSO₄ and the solvent removed by rotaryevaporation. The crude product was purified by column chromatographyusing 1:1 hexanes: EtOAc as eluent. Yield=0.38 g, 27%. ¹H NMR (CDCl₃) δ0.98, m, 6H; 1.31-1.77, m, 8H; 2.73, br t J=7.5 Hz, 1H; 3.29, td J=6.9,2.1 Hz, 1H; 6.48, br s, 1H. ¹³C NMR (CDCl₃) δ 14.11, 14.16, 19.90,20.78, 30.89, 37.47, 55.38, 56.91, 171.83. MS-EI: m/z=156.2 [M+H]⁺.

Synthesis of Compound (15):

Compound 15 was prepared using a modified literature procedure. SeeParsons, P. J.; Camp, N. P.; Underwood, J. M.; Harvey, D. M. (1996)“Tandem Reactions of Anions: A Short and Efficient Route to±Anatoxin-a.” Tetrahedron 52: 11637-11642. In a 50 mL round-bottomedflask was combined 4-tert-butylstyrene (0.016 mol, 2.62 g) and drydiethyl ether (5 mL). The mixture was cooled to 0° C. and stirred underN₂. Chlorosulfonyl isocyanate (CSI) (1 eq., 0.016 mol, 2.32 g) was addeddropwise to the cooled reaction mixture. The reaction was allowed tostir at 0° C. for 1 h and then warned to room temperature overnight. Thereaction was then diluted with chloroform (20 mL), cooled to 0° C. andquenched by addition into a stirring aqueous solution (100 mL) of Na₂SO₃(12 g) and Na₂PO₄ (14 g), keeping the temperature below 25° C. and thepH between 6 and 8 by additions of 2 M NaOH. The reaction was allowed towarm to room temperature overnight. The layers were separated and theaqueous portion was extracted with chloroform. The combined organicportions were dried over MgSO₄ and the solvent removed by rotaryevaporation. The crude product was recrystallized from diethyl ether.Yield: 1.8 g, 54%. ¹H NMR (300 MHz, CDCl₃, ppm) δ 1.32, s, 9H; 2.90, dddJ=15, 2.5, 1.1 Hz, 1H; 3.43, ddd J=15, 5.1, 2.4 Hz, 1H; 4.70, dd J=5.4,2.7 Hz, 1H; 6.1, s, 1H; 7.37, app. dd J=33, 10.8 Hz, 4H. MS (ESI)=429.5[2M+Na]⁺.

Example 2—Polymer Synthesis

Materials:

All reagents were obtained from Aldrich (Milwaukee, Wis.) and used asreceived. CH₂Cl₂ and THF were distilled under reduced pressure overCaH₂.

Instrumentation:

¹H (300 MHz) and ¹³C (75 MHz) NMR spectra were obtained on a BrukerAC+300 NMR spectrometer. Gel Permeation Chromatography (GPC) wasperformed using a Shimadzu LC-10AD liquid chromatography (HPLC) pumpequipped with Wyatt miniDawn and Optilab rex detectors. The mobile phasewas THF with a flow rate of 1 mL/min. Separations were performed usingTSK-GEL column set (2×GMH_(HR)-H).

Homopolymerization of 6:

Polymerization of 6 is a representative procedure for thepolymerizations of β-lactam monomers 3-7, 10, and 13-15. Anyexperimental and observational exceptions will be noted. In a 7 mL glassvial, under inert atmosphere, was combined 6 (1 mmol, 153 mg), potassiumtert-butoxide (KOtBu, 0.045 mmol, 5 mg) as base to deprotonate a certainfraction of the monomer, and 2 (0.02 mmol, 5 mg) as coinitiator and asthe means to control the molecular weight. Monomer to coinitiator ratiosranging from 1/10 to 1/250 were successfully employed depending on thetargeted molecular weight. The mixture was dissolved by addition ofdichloromethane (CH₂Cl₂, 1 mL), or THF (1 mL) and kept under roomtemperature for 0.5 to 4 hours depending on the monomer to coinitiatorratio where higher ratios require longer polymerization times. Then themixture was opened to air, the polymer was precipitated into pentane (10mL), and isolated by centrifuging and removing the supernatant. Polymerwas dried overnight under reduced pressure at room temperature. Theisolated yield was 95% (146 mg). ¹H NMR (300 MHz, CDCl₃, ppm) δ1.10-2.10, broad s, 12H; 2.15-3.10, broad m, 1H; 4.2-5.0, broad m, 1H;7.43, m, end-group low-resolution peak; 7.89, m, end-grouplow-resolution peak. M_(n)=5840 g/mol, polydispersity index (PDI)=1.02(dn/dc=1.37).

Alternative Compounds as Base Initiator:

The general procedure described in the above paragraph was employed byreplacing KOtBu with an alternative base including lithiumbis(trimethylsilyl)amide (LiN(TMS)₂), potassium bis(trimethylsilyl)amide(KN(TMS)₂, sodium methoxide (NaOEt, in tetrahydrofuran). All of theabove mentioned bases resulted in low PDI polymers with molecularweights in close approximation to targeted molecular weights.

Less Preferred Initiators:

The following metal complexes Sc(N(TMS)₂)₃, Al₂(N(Me)₂)₆, Al(N(TMS)₂)₃,Zn(N(TMS)₂)₂, Sn(N(TMS)₂)₂, and CpTi(N(Me)₂)₂Cl were employed in theabove described general polymerization procedure and initial resultsshowed the reactions resulted in the recovery of more than 90 mol % ofthe monomers. Little polymeric product was obtained. Thus, these metalcomplexes are not preferred for use in the present invention.

Alternative Homopolymerization of 6:

Here, the reaction takes place in THF at ambient temperature in thepresence of a base [LiN(SiMe₃)₂] and a co-initiator (as shown either4-tert-butyl benzoyl chloride (TBBC) or compound 2). The appropriateamounts of the monomer and co-initiator were mixed together in THF,whereupon a solution of base was added to the mixture in one portion.After 1 hour, the polymer product was precipitated by adding pentane tothe reaction solution. The product was isolated by centrifugation anddried under high vacuum. 4-Tert-butyl benzoyl chloride or theN-benzoyl-β-lactam (compound 2) were used as co-initiators.

Reactions using 4-tert-butyl benzoyl chloride as the co-initiator showednarrower polydispersity of the polymer product obtained as compared toreactions using compound 2 as the co-initiator. Other bases (KOtBu, KH,NaOMe, NaOH) and alternative solvents and solvent mixtures (Cl₂Cl₂,MeOH, THP/H₂O) were also tried for this reaction. Polymerization readilyoccurred in CH₂Cl₂, but the resulting polymer had slightly broaderpolydispersity. Similar increased broadening of the polydispersity wasobserved when KOtBu and NaOMe(THF) were used as the base. The use ofheterogeneous KH as a base yielded a polymer with PDI>2. Adding water oramine (20 mol %) to the reaction yields a polymer product with arelatively small molecular weight, but without any broadening of the PDIof the product (<2). Both water and amine are known to poison otheranionic polymerization reactions. Polydispersity broadened (from about1.06 to 1.27) when the polymerization reaction is allowed to proceed forseveral hours.

A host of monomer, co-initiator, solvent, and base combinations werefabricated in the same fashion as reported here. The results aresummarized in Table 2.

TABLE 2 Polymerization results for monomers 1-5([monomer]/[co-initiator] = 30): Monomer Co-initiator Base SolventYield, % M_(n) PDI 6 TBBC LiN(SiMe₃)₂ THF 96 4,900 1.05 6 Cmpd 2LiN(SiMe₃)₂ THF 95 4,700 1.12 6 TBBC LiN(SiMe₃)₂ CH₂Cl₂ 93 5,100 1.14 6TBBC KOtBu THF 96 5,400 1.11 6 Cmpd 2 KOtBu CH₂Cl₂ 95 4,900 1.15 6 TBBCKH THF 94 6,800 2.40 6 TBBC MeONa THF 97 5,100 1.14 6 TBBC MeONa MeOH Noreaction 6 Cmpd 2 NaOH THF/H₂O (1:1) No reaction 6 Cmpd 2 LiN(SiMe₃)₂THF/BnNH₂ 92 5,200 1.12 (20% mol to monomer) 6 Cmpd 2 LiN(SiMe₃)₂THF/H₂O 90 4,950 1.14 (20% mol to monomer) 6 Cmpd 2 LiN(SiMe₃)₂ THF/H₂O(1:20) 93 4,500 1.16 5 TBBC LiN(SiMe₃)₂ THF 97 Insoluble in THF 7 TBBCLiN(SiMe₃)₂ THF 95 7,300 1.10 10 TBBC LiN(SiMe₃)₂ THF 98 5,200 1.06 13TBBC LiN(SiMe₃)₂ THF 98 5,900 1.10This Example demonstrates a general approach for preparingβ-polypeptides bearing side chains having polar functional groups.Homopolymerization of Other β-Lactam-Containing Monomers:

Using the approach recited in the immediately prior Example, thefollowing bicyclic and monocyclic monomers were also successfullypolymerized:

Homopolymerization of 3:

In a 7 mL glass vial, under inert atmosphere, was combined 3 (0.2 mmol,30 mg), potassium tert-butoxide (KOtBu, 0.0045 mmol, 0.5 mg), and 2(0.002 mmol, 0.5 mg). The polymer was isolated as described above forpoly(6). The isolated yield was 92% (142 mg). ¹H NMR (300 MHz, CDCl₃,ppm) δ 1.30-3.20, broad m, 9H; 4.20-4.80, broad s, 1H; 5.40-5.80, broads, 2H; 7.43, m, end-group low-resolution peak; 7.89, m, end-grouplow-resolution peak. M_(n)=16.000 g/mol, PDI=1.2 (for dn/dc=1.37). TheGPC curve for this polymer is shown in FIG. 1.

Homopolymerization of 5:

The isolated yield was 90% (139 mg). ¹H NMR (300 MHz, CDCl₃, ppm) δ1.10-2.05, broad m, 8H; 2.53, s, 1H; 3.98, s, 1H; 7.43, m, end-grouplow-resolution peak: 7.89, m, end-group low-resolution peak. Theresulting polymer was mostly insoluble in THF.

Homopolymerization of 10:

In a 7 mL glass vial, under inert atmosphere, was combined 10 (0.2 mmol,45 mg), potassium tert-butoxide (KOtBu, 0.02 mmol, 2 mg), and 2 (0.014mmol, 3.5 mg). The polymer was isolated as described above for poly(6).The isolated yield was 95% (44 mg). ¹H NMR (300 MHz, CDCl₃, ppm) δ1.00-1.80, broad s, 9H; 2.05-3.70, overlapping resonances, broad m, 9H;7.50, m, end-group low-resolution peak; 7.94, m, end-grouplow-resolution peak. M_(n)=10,400 g/mol. PDI=1.16 (for dn/dc=1.37). Thet-BOC protected primary amine groups on this polymer were deprotected bydissolution of the polymer in trifluoroacetic acid (100 mg/mL) andtreating at 55° C. for 8 hours, resulting in a water soluble polymer. ¹HNMR (300 MHz, D₂O, ppm) δ 1.10-1.70, m, 6H; 2.9-3.6, broad overlappingpeak, 3.19, s, 1H; 3.40, s, 1H; 7.42-7-71, m, end-group low-resolutionpeak.

Homopolymerization of 13:

In a 7 mL glass vial, under inert atmosphere, was combined 10 (0.14mmol, 30 mg), potassium tert-butoxide (KOtBu, 0.01 mmol, 1 mg), and 2(0.004 mmol, 1 mg). The polymer was isolated as described above forpoly(6). The isolated yield was 91% (27 mg). ¹H NMR (300 MHz, CDCl₃,ppm) δ 1.18, broad s, 3H; 1.43, broad s, 9H; 2.2, s, 1H, 2.4-4.4, set ofoverlapped resonances, 4H; 7.50, m, end-group low-resolution peak; 7.94,m, end-group low-resolution peak. Deprotected water soluble poly(13) wasobtained as described for poly(10). ¹H NMR (300 MHz, D₂O, ppm) δ 1.11,s, 3H; 2.9, m, 2H; 3.22, s, 1H; 4.17, s, 1H; 7.35-7-62, m, end-grouplow-resolution peak.

Homopolymerization of 14:

When the general polymerization procedure is applied to 14, thepolymerization mixture solidifies within 5 minutes. Polymer isextensively washed with ether resulting in a white powder, insoluble inchloroform, THF, and DMSO.

Homopolymerization of 15:

When the general polymerization procedure is applied to 14, thepolymerization mixture stays homogeneous. However when polymer isprecipitated in pentane the resulting white powder is insoluble inchloroform, THF, and DMSO.

Example 3—Living Polymerization

(a) Molecular Weight of the Product Polymer as a Function of the Ratioof Monomer-to-Co-Initiator Ratio ([Monomer]/[Co-Initiator]):

A study was conducted to determine if systematically adjusting the ratioof the concentration of monomer reactants to the concentration of theco-initiator ([monomer]/[co-initiator]) would have a correspondingeffect on the molecular weight of the resulting polymer product.Compound 6 was used as the monomer for this Example. The results aredepicted in FIG. 3, which is a graph depicting [monomer]/[co-initiator]on the X-axis and product molecular weight (Mn) on the Y-axis. As can beseen from FIG. 3, the molecular weight of the polymers obtained versusthe [monomer]/[co-initiator] ratio shows a linear, proportionaldependence up to about [monomer]/[co-initiator]=80. These resultsreflect and confirm the living character of the polymerization reaction.

(b) Degenerate Block Co-Polymerization:

The living character of polymerization was also confirmed by carryingout degenerate block-copolymerization of monomer 6 as shown in ReactionScheme 6:

The GPC curves, shown in FIGS. 4 and 5, show the complete consumption ofthe initial polymer product A (FIG. 4) and the appearance of adegenerate monomodal block-copolymer product B (FIG. 5). Polymerizationof compound 6 with MeONa and compound 5 with LiN(SiMe₃)₂ were alsoconfirmed to proceed in living fashion. This is significant because itallows for exquisite control of the product using homopolymerization orcopolymerization.

Example 4—Random Copolymerizations

The general polymerization procedure was applied to mixtures of monomersresulting in polymers without any broadening in molecular weightdistributions. ¹H NMR analysis showed the presence of all resonancesfrom individual homopolymers overlapped.

Example 5—Block Copolymerizations

Block copolymers were prepared by sequential comonomer addition. First adesired homopolymer was prepared according to the general polymerizationprocedures recited above, and after allowing time for the completion offirst block (20 to 120 minutes depending on monomer to initiator ratios)a second monomer was added and second block was formed. Alternatively ahomopolymer can be isolated by precipitation, redissolved in apolymerization mixture according to the general polymerization procedureas a replacement for coinitiator 2, and lead to growth of a second blockfrom the chain-end of the first block. Comparisons of molecular weightsof first block and diblock showed the expected increase in molecularweight without a significant broadening in molecular weightdistributions. In particular, see FIG. 2, which shows two superimposedGPC curves for a homopolymer and a diblock co-polymer fabricated usingthe homopolymer. The earlier eluting peak in FIG. 2 is the diblockcopolymer, and the later eluting peak is the homopolymer.

Example 6—Terminal Functionalization

Because the polymerization proceeds in a living fashion, the termini ofthe polymer chains can be functionalized using appropriate co-initiatorsthat also function as a terminal co-monomer reactant. Co-monomers forterminal functionalization can include broad classes of functionalgroups, including hydrophobic, anionic, cationic and neutral hydrophilicgroups, without limitation. An attractive feature of the polymerizationmethod is the ability to control the functional groups located at eitherend of the polymer. The acylating agent used as the co-initiator (incombination with a strong base) functionalizes one end of the polymerchain and the other end of the chain possesses an imide group that willreact with suitable nucleophiles (e.g., primary amines) after thepolymerization reaction is complete. By exploiting the fundamentalcharacteristics of the inventive polymerization technique, one canintroduce a wide range of functional groups into the polymer, both alongthe main chain (via side chains incorporated into the monomers) and ateach end of the polymer (by choosing an appropriate co-initiator andreaction the terminal imide group after polymerization is complete).These functional groups are expected to play a significant role in thebiological activity of the material.

In this Example, 4-chloromethyl benzoyl chloride (C) was used as aco-initiator to yield a polymer having a 4-chloromethyl benzoylterminus. See Reaction Scheme 7:

Further chemical modification of the 4-chloromethyl end-group yielded ahost of end-group functionalized β-polypeptide polymer derivatives asshown in Reaction Scheme 8:

As shown in Reaction Scheme 8, the reactive 4-chloromethyl group can beused to append various functional end groups in high yields, such asaldehyde, esters, thioesters, amines and imides (phthalimide followed bydeprotection), and the like.

An α,β-unsaturated carbonyl terminus can be appended to the polymerchain by running the reaction using an appropriate co-initiator, asshown in Reaction Scheme 9:

As in the case of Reaction Scheme 8, the terminal methylene group canfunction as a reactive site to allow for further modification of thepolymer chain.

Example 7—Measurement for Antibacterial Activities of Poly-β-Peptides

The bacteria strains used in these assays were Escherichia coli JM109,Bacillus subtilis BR151, Staphylococcus aureus 1206(methicillin-resistant), and Enterococcus faecium A634(vancomycin-resistant). The antibacterial activity for thepoly-b-peptides was determined in sterile 96-well plates (Falcon 3075microtiter plate) by a microdilution method. A bacterial suspension ofapproximately 10⁶ CFU/MI in BHI medium was added in 50 μL aliquots to 50μL of medium containing the poly-β-peptides in 2-fold serial dilutionsfor a total volume of 100 μL in each well. The plates were incubated at37° C. for 6 hours. Growth inhibition was determined by measuring the ODat wavelengths ranging from 595-650 nm. Each MIC is the result of atleast two separate trials: each trial is the result of an assay run induplicate. MIC determinations were reproducible to within a factor oftwo and are reported as the highest (most conservative) of thedetermined values.

Example 8—Measurement for Hemolytic Activities of Poly-β-Peptides

Freshly drawn human red blood cells (hRBC, blood type A) were washedseveral times with Tris buffer (pH 7.2, 150 mM NaCl) and centrifuged at2000×rpm until the supernatant was clear. Two-fold dilutions ofpoly-β-peptides in Tris buffer (pH 7.2, 150 mM NaCl) were added to eachwell in a sterile 96-well plate (Falcon 3075 microtiter plate), for atotal volume of 20 μL in each well. A 1% v/v hRBC suspension (80 μL inTris buffer) was added to each well. The plate was incubated at 37° C.for 1 hour and then the cells were pelleted by centrifugation at 3500rpm for 5 minutes. The supernatant (80 μL) was diluted with Milliporewater (80 μL), and hemoglobin was detected by measuring the OD at 405nm. The OD of cells incubated with mellitin at 400 μg/ml defines 100%;the OD of cells incubated in Tris buffer defines 0%.

What is claimed is:
 1. A β-polypeptide comprising internal subunits having a structure selected from the group consisting of:

wherein: R³, R⁴, R⁵, and R⁶ are each independently selected from the group consisting of hydrogen, substituted or unsubstituted C₁-C₆-alkyl, aryl, C₁-C₆-alkylaryl, amino, protected-amino, amino-C₁-C₆-alkyl, and protected-amino-C₁-C₆-alkyl; A together with the carbon atoms to which it is attached is selected from the group consisting of substituted or unsubstituted C₅-C₁₂ cycloalkyl, C₅-C₁₂ cycloalkenyl, and five- to twelve-membered heterocyclic; the β-polypeptide has a molecular weight (M_(n)) of from 3,300 Da to 20,000 Da; and the β-polypeptide comprises heterochiral internal subunits.
 2. The β-polypeptide of claim 1, wherein the β-polypeptide has a molecular weight (M_(n)) of from 4,950 Da to 20,000 Da.
 3. The β-polypeptide of claim 1, wherein the β-polypeptide comprises a terminal subunit having a structure of:

wherein R is selected from the group consisting of linear, branched, or cyclic alkyl, alkenyl, alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted arylalkyl.
 4. The β-polypeptide of claim 3, wherein R in the terminal subunit is:

wherein R⁷ is selected from the group consisting of tert-butyl, chloromethyl, aldehyde, an ester, a thioester, an amine, and an imide.
 5. The β-polypeptide of claim 3, wherein R in the terminal subunit is selected from the group consisting of:


6. The β-polypeptide of claim 3, wherein the β-polypeptide comprises a second terminal subunit selected from the group consisting of:

wherein R³, R⁴, R⁵, R⁶, and A are as previously defined.
 7. The β-polypeptide of claim 6, wherein at least one of R³, R⁴, R⁵, and R⁶ in the second terminal subunit is selected from the group consisting of amino, protected-amino, amino-C₁-C₆-alkyl, and protected-amino-C₁-C₆-alkyl.
 8. The β-polypeptide of claim 1, wherein at least one of the internal subunits has a structure of:

wherein A is as previously defined.
 9. The β-polypeptide of claim 8, wherein A is selected from the group consisting of substituted or unsubstituted cyclohexane, cyclooctane, cyclooctene, and dodecane.
 10. A β-polypeptide comprising internal subunits having a structure selected from the group consisting of:

wherein: R³, R⁴, R⁵, and R⁶ are each independently selected from the group consisting of hydrogen, substituted or unsubstituted C₁-C₆-alkyl, aryl, C₁-C₆-alkylaryl, amino, protected-amino, amino-C₁-C₆-alkyl, and protected-amino-C₁-C₆-alkyl; A together with the carbon atoms to which it is attached is selected from the group consisting of substituted or unsubstituted C₅-C₁₂ cycloalkyl, C₅-C₁₂ cycloalkenyl, and five- to twelve-membered heterocyclic; the β-polypeptide has a molecular weight (M_(n)) of from 4,950 Da to 20,000 Da; and at least one of the internal subunits has a structure of:

wherein at least one of R³, R⁴, R⁵, and R⁶ in the at least one of the internal subunits is selected from the group consisting of amino, protected-amino, amino-C₁-C₆-alkyl, and protected-amino-C₁-C₆-alkyl.
 11. The β-polypeptide of claim 10, wherein the β-polypeptide comprises heterochiral internal subunits.
 12. A β-polypeptide comprising internal subunits having a structure selected from the group consisting of:

wherein: R³, R⁴, R⁵, and R⁶ are each independently selected from the group consisting of hydrogen, substituted or unsubstituted C₁-C₆-alkyl, aryl, C₁-C₆-alkylaryl, amino, protected-amino, amino-C₁-C₆-alkyl, and protected-amino-C₁-C₆-alkyl; A together with the carbon atoms to which it is attached is selected from the group consisting of substituted or unsubstituted C₅-C₁₂ cycloalkyl, C₅-C₁₂ cycloalkenyl, and five- to twelve-membered heterocyclic; and the β-polypeptide comprises heterochiral internal subunits.
 13. The β-polypeptide of claim 12, wherein the β-polypeptide has a molecular weight (M_(n)) of from 1,180 Da to 20,000 Da.
 14. The β-polypeptide of claim 12, wherein the β-polypeptide has a molecular weight (M_(n)) of from 7,300 Da to 20,000 Da.
 15. The β-polypeptide of claim 12, wherein the β-polypeptide comprises a terminal subunit having a structure of:

wherein R is selected from the group consisting of linear, branched, or cyclic alkyl, alkenyl, alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted arylalkyl.
 16. The β-polypeptide of claim 15, wherein the β-polypeptide has a molecular weight (M_(n)) of from 3,300 Da to 20,000 Da.
 17. The β-polypeptide of claim 15, wherein R in the terminal subunit is:

wherein R⁷ is selected from the group consisting of tert-butyl, chloromethyl, aldehyde, an ester, a thioester, an amine, and an imide.
 18. The β-polypeptide of claim 15, wherein R in the terminal subunit is selected from the group consisting of:


19. The β-polypeptide of claim 15, wherein the β-polypeptide comprises a second terminal subunit selected from the group consisting of:

wherein R³, R⁴, R⁵, R⁶, and A are as previously defined.
 20. The β-polypeptide of claim 19, wherein at least one of R³, R⁴, R⁵, and R⁶ in the second terminal subunit is selected from the group consisting of amino, protected-amino, amino-C₁-C₆-alkyl, and protected-amino-C₁-C₆-alkyl.
 21. The β-polypeptide of claim 12, wherein the β-polypeptide comprises a terminal subunit selected from the group consisting of:

wherein R³, R⁴, R⁵, R⁶, and A are as previously defined.
 22. The β-polypeptide of claim 21, wherein at least one of R³, R⁴, R⁵, and R⁶ in the terminal subunit is selected from the group consisting of amino, protected-amino, amino-C₁-C₆-alkyl, and protected-amino-C₁-C₆-alkyl.
 23. The β-polypeptide of claim 12, wherein at least one of the internal subunits has a structure of:

wherein at least one of R³, R⁴, R⁵, and R⁶ in the at least one of the internal subunits is selected from the group consisting of amino, protected-amino, amino-C₁-C₆-alkyl, and protected-amino-C₁-C₆-alkyl.
 24. The β-polypeptide of claim 12, wherein at least one of the internal subunits has a structure of:

wherein A is as previously defined.
 25. The β-polypeptide of claim 24, wherein A is selected from the group consisting of substituted or unsubstituted cyclohexane, cyclooctane, cyclooctene, and dodecane. 